Construction of a plasmid interspecific transfer system in Bacillus species with the counter-selectable marker mazF.

Bacillus sp. strains as attractive hosts for the production of heterologous secretory proteins usually play important roles in bio-industry. However, low transformation efficiency of exogenous plasmids limited the application of Bacillus species. Here, a novel plasmid interspecific transfer system, with high transformation efficiency, high positive rate, and convenient manipulation, has been successfully constructed. A high electrotransformation efficiency strain Bacillus subtilis F-168 containing the counter-selectable marker mazF was used as the plasmid donor strain in this transfer method. A shuttled plasmid pBE980 and its recombinant plasmids pBE980::pulA and pBE980::HSPA were successfully transferred into the recipient Bacillus strains (Bacillus amyloliquefaciens 66, Bacillus licheniformis 124 and Bacillus megaterium 258) by this method. After co-culturing the donor cells (OD600nm = 1.3-1.7) and the recipient cells (OD600nm = 0.5-0.9) for 24 h in 22 °C, more than 1.0 × 105 positive transformants were obtained and a interspecific transformation efficiency of 1.0 × 10-3. It would provide a new approach for genetic manipulation in Bacillus strains and accelerate the research progress of the wild Bacillus strains in bio-industry.